Confocal microscopy is well known in the art. The concept of confocal microscopy is that the image viewed by the microscope is confined to a very precise focal plane by limiting the depth of field of the image. Only those portions of the specimen which are in focus are detected. Out of focus regions of the sample appear dark. By changing the position of the focal plane, this important principle defines one major method for achieving optical sectioning.
The earliest versions of confocal microscopes used direct vision design with incoherent illumination. The field of illumination in the specimen was limited by a pin hole positioned on the illumination axis. The image of this pin hole is then projected on the specimen by a condenser lens. The illuminated point on the specimen reflects light (or as described below, emits fluorescent light). The reflected light of the image is then focused through an objective lens onto a detector. Either the specimen or the light focused on the image is scanned in a raster pattern so that the detector collects pixel information from a region of the specimen. The pixel information is then passed through a computer which can generate an image of the overall specimen.
The concept of the scanning confocal microscope is described in U.S. Pat. No. 3,013,467 to Marvin Minsky, which is hereby incorporated by reference. The optical path of the scanning confocal microscope may be constructed in transilluminating mode in which a separate condenser and objective lens is used in the same axis. In the alternative, the optical path of the scanning confocal microscope may be constructed in an epi-illuminating mode making a single objective lens serve both as the condenser and objective lens and using a dichroic or half mirror to collect the emitted light into a detector, as shown in FIG. 1.
In the Minsky patent, the raster scan is generated by moving the stage on which the specimen is supported by two orthogonally vibrating tuning forks that are driven by electromagnets. As the stage is moved in a raster scan pattern, the resulting image detected by the image detector is a serial raster scanned image.
The use of fluorescent dyes to stain the specimen being viewed further improved the range of applications to which scanning confocal microscopy could be applied. Especially in the area of immunofluorescence histochemistry and in other neuroanatomical techniques, the staining of specimens with dyes is particularly useful to aid in distinguishing different features within biological tissues. The stains may comprise dyes designed only to absorb light or dyes that emit light in response to absorption, which is called fluorescence. Fluorescent dyes have the advantage over dyes which only absorb light in that a given fluorescent marker will be visible only when illuminated with the appropriate filter set.
Fluorescence is a consequence of the interaction of a photon with a fluorophore. When a photon of light is absorbed by a molecule it may increase the potential energy of the molecule by raising an electron to a higher orbital state. An electron raised to a higher orbital state from its natural state will tend to revert to the natural state. When the electron falls from a higher to a lower orbital state, energy is released which is equal to the difference in energy between the two orbital states. When this occurs, part or all of its energy may be released as a photon having a wavelength (spectral line) proportional to the released energy. The resulting luminescence is called fluorescence (and in some circumstances phosphorescence). Excitation of a fluorophore molecule at one wavelength typically results in fluorescent emission at longer wavelengths of light.
The scanning laser confocal microscope improved on the design of the scanning confocal microscope and the use of fluorophores by using coherent light to scan the stained specimen. The monochromaticity, high intensity and lack of divergence of the laser light contributed to improvements in the resulting images. In an epi-illuminating laser scanning confocal microscope of the prior art, as shown in FIG. 2, the laser light 200 is scanned onto the specimen 220 from above and is reflected to a detector 215 in the same focal path as the incident light through the use of a half mirror or dichroic mirror 205. Typically, the specimen is stained with fluorescent dyes to enhance specific features within the specimen which may be of interest.
The MRC-600 laser scanning confocal imaging system, shown diagrammatically in FIG. 2, is manufactured by BioRad Microscience of Hemel Hempstead, Herts, England. This laser scanning confocal microscope system is adaptable for use with a number of upright and inverted microscopes available from microscope vendors such as Nikon, Zeiss, Olympus and Leitz. The coherent illumination is an argon ion laser 201 having primary lines at 514 and 488 nanometers (nm). The emitted laser light 200 also has a plurality of minor spectral lines as determined by argon. The lines are filtered by the external filter 203 which selects either the 488 nm (blue) line or the 514 nm (green) line by means of an excitation filter 203. The selected light is reflected by a beam splitter 205 which includes a dichroic mirror used for fluorescent imaging. If simple reflection imaging is required, a semi-reflecting or half mirror may be used in place of the dichroic mirror 205.
The argon ion laser is available from Ion Laser Technology Company of Salt Lake City, Utah, part number 5425A. The argon ion laser is used to excite fluorescent dyes in the specimen which emit light slightly shifted in the spectrum in response to the excitation wavelength of the spectral lines of the laser light. The dyes are selected based upon their sensitivity to light, their affinity for features desired to be viewed in specimens and their fluorescent capabilities.
The light 200 from the laser is passed through the scanning unit 207 where it is raster scanned in an XY scanning movement by means of two oscillating mirrors. The laser beam is then passed through a microscope eyepiece onto the specimen such that a scanning spot caused by the scanning unit 207 scans the specimen. Reflected light or fluorescent light from the specimen passes back through the scanning system along the same path as the incident laser light. Reflection of the light is so rapid that the mirrors have not shifted position so that the light retraces the exact original path in the reverse direction. A portion of the reflected or fluorescent light passes through a half mirror or dichroic mirror 205 to be passed to photomultiplier tubes.
The laser scanning confocal imaging system from BioRad shown in FIG. 2 attempts simultaneous imaging of two different fluorescent stains. The 514 nm spectral line from argon ion laser 201 is used to excite both fluorescein isothiocyanate (fluorescein) and Texas Red.TM. (from Molecular Probes, Inc.) conjugated probes. This attempts the simultaneous excitation of different fluorescent dyes to allow selected features of the specimen to be stained in different colors and viewed together. The dual images are picked up by photomultiplier tubes 213 and 215. A second beam splitter 208 is a dichroic mirror allowing light of one wavelength to be directed to photomultiplier tube 213 while light having other wavelengths passed to photomultiplier 215.
The two images received from photomultiplier tubes 213 and 215 are used by a computer 222 to construct an image on display 224 of the specimen in a single focal plane. The simultaneous imaging of two different fluorescent stains at exactly the same focal plane would allow the identification of different specific features in the same specimen. A shortcoming of the dual color laser scanning confocal microscope system of the prior art is that the 514 nm line of the argon ion laser produces simultaneous excitation of the two fluorescent dyes (fluorescein and Texas Red.TM.). This simultaneous excitation causes false imaging and the loss of feature detail in the resulting image generated by the computer.
FIG. 3 shows a graph published by BioRad Microscience indicating the absorption and emission spectra of fluorescein and Texas Red.TM.. The graph is reproduced from BioRad and only approximates the spectrums. Curve 301 describes the absorption spectra of fluorescein while curve 303 shows the emission spectra of fluorescein. Curve 305 shows the absorption spectra of Texas Red.TM. and curve 307 describes the emission spectra of Texas Red.TM.. As can be seen in FIG. 3, there is an area of overlap between the absorption spectrums of Texas Red.TM. and fluorescein at 514 nm. Thus, simultaneous excitation and emission of fluorescein and Texas Red.TM. occurs when excited with the single 514 nm line of the argon laser. Also shown in FIG. 3 is a large area of overlap between the emission spectra of Texas Red.TM. and the emission spectra of fluorescein.
The response curves for the filters and the dichroic reflectors are placed below the absorption and emission spectra of fluorescein and Texas Red.TM. in FIG. 3 for comparison. When using an argon laser to excite the dyes, the 514 nm line of the dye is the only line allowed to pass through the exciter filter 203 shown in FIG. 2. The narrow wavelength response curve 309 of FIG. 3 is for the exciter filter 203. The response curve 311 is for dichroic reflector 205 and the response curve 313 is for dichroic reflector 208. The response curve 315 is for green channel filter 211 and the response curve 317 is for red channel filter 209.
As can be seen in FIGS. 3, the intent is to have the single 514 nm line of the argon laser excite both the fluorescein and Texas Red.TM. dyes. The emission spectra of these respective dyes are then selected to be passed to photomultiplier tubes 213 and 215 shown in FIG. 2 to be independently detected for reconstructing a two color image at the same focal plane. The problem with this prior art technique is that the single excitation line from the argon laser excites fluorescein much more efficiently than Texas Red. For example, as shown in FIG. 3, the excitation of the fluorescein dye at a wavelength of 514 nm is at approximately 50%. The excitation of Texas Red.TM. at the same 514 nm wavelength, however, is very low (less than 3%). Since the emission spectra of the dyes corresponds to, and is proportional to the amount of energy absorbed by the dyes, the low amount of absorbed energy from the 514 nm line by Texas Red.TM. will result in a very low amount of emitted fluorescent light. Hence, the amount of fluorescein emission seen in the red channel can vary according to the relative concentrations of fluorescein and Texas Red.TM.. Unless the relative concentrations and saturation of the dyes accurately controlled, the emission spectra of Texas Red.TM. may be swamped by the "spillover" of the longer wavelengths of the fluorescein emission spectra. This confusion will result in images in which many of the features stained only with fluorescein will appear in both images. One solution to this problem is to use separate laser lines to better excite both fluorescent dyes.
Multiple line excitation of specimens dyed with different fluorochromes using two lasers is also known in the prior art. For example, a Spectra-Physics 2025 argon ion 3-watt water cooled laser (tunable to a single argon ion line between 351 nm through 528 nm) has been confocally aligned with a 5 milliwatt air-cooled argon ion laser having fixed wavelengths at 488 nm and 514 nm. The alignment of two lasers, however, presents extreme focusing problems. The two light paths must be aligned to exacting standards to ensure that the same focal plane is observed.